Methods for evaluating cell proliferation are important for research in the field of developmental biology, cancer biology and cell biology. Recent years, Click-iT EdU Assays by Invitrogen is gaining popularity due to its fast staining speed, mild tissue treatment condition, reproducibility and specificity. However, the standard kit from Invitrogen is designed for tissue cultured cells, hence the manufactory provided protocol is optimized for cells cultured on coverslips. EdU staining became a popular method to evaluate cell proliferation in vivo in zebrafish embryos. However, the protocols that we have tested in the past often yield inconsistent results due to variable EdU incorporation efficiency. Here we describe an EdU staining protocol that we optimized for zebrafish larvae, which incorporated a crucial EdU injection step. Our protocol was used successfully by many new students in training, without any issues.